Rat brain protein kinase C. Kinetic analysis of substrate dependence, allosteric regulation, and autophosphorylation.
Kinetic studies on the interaction of protein kinase C with cations and substrates were performed and the effects of essential activators on the interaction of protein kinase C with its substrates were studied. The catalytic fragment of protein kinase C interacted with protein substrate, MgATP, and Mg2+. The dual divalent cation requirement was shown by kinetic analysis as well as by the ability of Mn2+ to substitute for Mg2+. Analysis of kinetic data based on equilibrium assumptions suggested a random order of interaction of the catalytic fragment with its substrate and Mg2+ cofactor. Activation of intact protein kinase C required Ca2+, phosphatidylserine (PS), and diacylglycerol (DAG) as essential activators. Kinetic analysis of the interaction of activators with substrates indicated that Ca2+ and PS acted to increase the activity of the enzyme without modulating the KM for MgATP; PS and Ca2+ significantly decreased the KM for histone. DAG, on the other hand, did not affect the KM for either MgATP or histone but dramatically enhanced the kcat of the enzyme. These studies allow kinetic distinction between the effects of PS and Ca2+ on the one hand and DAG on the other. The possible interference of the kinetic analysis by histone was also examined by studying the requirements for autophosphorylation of protein kinase C; autophosphorylation showed similar dependencies on PS and DAG. There were no effects of histone on the lipid dependence of protein kinase C autophosphorylation, phorbol dibutyrate binding, and inhibition of autophosphorylation by sphingosine. These studies are discussed in relation to a kinetic model of protein kinase C activation.
Hannun
Y A
YA
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Bell
R M
RM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D001921:Brain / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008823:Micelles; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats
D008823:Micelles; D015240:Phorbol 12,13-Dibutyrate; D008345:Manganese; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D008274:Magnesium